J Korean Cancer Assoc.  1998 Oct;30(5):997-1004.

Role of AP-1 Transcriptional Regulation of Vimentin Gene during 12-O-Tetradecanoylphorbol-13-Acetate Differentiation of HL-60 Cells

Affiliations
  • 1Department of Biochemistry, Chungnam National University, Daejeon, Korea.
  • 2Department of Surgery, College of Medicine, Chungnam National University, Daejeon, Korea.
  • 3Department of Cancer Research Institute, Chungnam National University, Daejeon, Korea.

Abstract

PURPOSE
To gain insight on the role of AP-1 in transcriptional regulation of vimentin gene during differentiation of HL-60 cells by 12-0-tetradecanoylphorbol-13-acetate (TPA), the levels of vimentin mRNA and AP-1 have been investigated with Northern blot hybridization and DNA mobility shift assay.
MATERIALS AND METHODS
HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics in a humidified 5% CO2 at 37 degree C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe(Upper strand, 5'-CGCTTGATGAGTCAGCCG- 3') for AP-1 binding activity was mixed with nuclear extracts in a 20 microliter reaction volume containing 300 mM KC1, 60 mM HEPES, pH 7.9, 25 mM MgCl2, 1 mM EDTA, 1 mM DTT, 60% glycerol, and 2 microgram of poly[dI-dC].
RESULTS
TPA increased vimentin mRNA levels, with maxima1 stimulation reached at 24 hr. The level of vimentin mRNA was induced in proportion to the concentration of TPA. TPA-induced vimentin mRNA was almost reduced by actinomycin-D pretreatment. TPA- induced stimulation of vimentin gene was completely reduced by staurosporin pretreatment. In DNA mobility shift assay, AP-I newly appeared at 24 hr during TPA- induced differentiation and was almost not detected after the pretreatment of staurosporin.
CONCLUSIONS
These results suggest that the induction of vimentin mRNA during TPA- dependent differentiation in HL-60 cells may be mediated by protein kinases C signal transduction and AP-1 is important to transcriptional regulation.

Keyword

Vimentin; AP-1; Staurosporin; TPA; HL-60

MeSH Terms

Anti-Bacterial Agents
Blotting, Northern
DNA
Edetic Acid
Electrophoretic Mobility Shift Assay
Glycerol
HEPES
HL-60 Cells*
Humans
Hydrogen-Ion Concentration
Magnesium Chloride
Protein Kinases
RNA
RNA, Messenger
Signal Transduction
Transcription Factor AP-1*
Vimentin*
Anti-Bacterial Agents
DNA
Edetic Acid
Glycerol
HEPES
Magnesium Chloride
Protein Kinases
RNA
RNA, Messenger
Transcription Factor AP-1
Vimentin
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